A peptidoglycan (PG) cell wall is an essential extracytoplasmic feature of most bacteria (Singer et al., 1989); this essentiality has made its biogenesis a fruitful target for antibiotics, including vancomycin and penicillin. The cell wall is directly exposed to the extracellular milieu in Gram-positive bacteria, but is shielded in Escherichia coli (E. coli) and other Gram-negative species by a highly selective permeability barrier formed by the outer membrane (OM). The OM prevents influx of antibiotics, such as vancomycin, restricting their access to intracellular targets (Eggert et al., 2001; Ruiz et al., 2005).
Lipopolysaccharide (LPS) forms the surface-exposed outer leaflet of the OM and is key to the barrier function (Osborn et al., 1972; Kamio and Nikaido, 1976; Nikaido, 2003). LPS is a glycolipid consisting of a ‘lipid A’ anchor within the bilayer, and a set of covalently attached distal ‘core’ saccharides (Raetz and Whitfield, 2002). LPS is made at the cytosolic leaflet of the inner membrane (IM) before being flipped to the periplasmic leaflet (Zhou et al., 1998). A transenvelope complex of seven lipopolysaccharide transport proteins (LptABCDEFG) delivers LPS from the IM to the OM (Ruiz et al., 2009; Chng, Gronenberg, et al., 2010). A sub-complex of the β-barrel LptD and lipoprotein LptE resides within the OM and accomplishes the final step of inserting LPS into the outer leaflet (Chng, Ruiz, et al., 2010).
LPS and PG are both potent activators of immune responses via distinct stimulatory mechanisms. However, LPS is inherently toxic to humans and animals due to hyper-activation of inflammatory immune responses. Detoxification can eliminate some or all of the endotoxicity, but the less toxic variants generally also have reduced immunostimulatory properties. LPS-stimulated immune responses can be synergistically increased by co-stimulation with PG added into a mixture (Fritz et al., 2005). Therefore, this synergy is likely to be improved by direct coupling of LPS and PG into a single molecule that allows both activators to stimulate their associated pathways. A detoxified LPS-PG molecule will likely retain more desirable immunostimulatory properties in comparison to detoxified LPS alone.